Liquid chromatography validation

A further thirty years were to pass before Kuhn and his co-workers (3) successfully repeated Tswetf s original work and separated lutein and xanthine from a plant extract. Nevertheless, despite the success of Kuhn et al and the validation of Tswett s experiments, the new technique attracted little interest and progress continued to be slow and desultory. In 1941 Martin and Synge (4) introduced liquid-liquid chromatography by supporting the stationary phase, in this case water, on silica in the form of a packed bed and used it to separate some acetyl amino acids. 

Section I of this book includes chapters on the principles and practice of PLC. After this introductory Chapter 1, Chapter 2 provides information on efforts undertaken to date in order to establish the theoretical foundations of PLC. With growing availability and popularity of modem computer-aided densitometers, separation results can be obtained in digital form as a series of concentration profiles that can be relatively easily assessed and processed. From these, relevant conclusions can be drawn in exactly the same manner as in automated column chromatographic techniques. Efforts undertaken to build a theoretical foundation of PLC largely consist of adaptation of known strategies (with their validity confirmed in preparative column liquid chromatography) to the working conditions of PLC systems. 

These authors noted the potential for the assay to underestimate the concentration of TSR due to decreased binding of metabolites relative to parent spinosad. However, the major residue found was parent spinosad, so underestimation of residues is not likely to be problematic. Overall, this method was validated in 34 matrices and showed excellent agreement with results obtained with a high-performance liquid chromatography/ultraviolet detection (HPLC/UV) method. ... 

Full acceptance of HPLC/MS methods by the US EPA OPP as enforcement methods occurred between 1998 and 2001. For example, in 1998, the EPA OPP accepted HPLC/MS (without MS/MS) methods as primary enforcement methods, and high-performance liquid chromatography/tandem mass spectrometry (HPLC/MS/MS) only was suitable for confirmatory methods. However, in 2001, HPLC/MS/MS methods also became acceptable for primary enforcement. Table 4 summarizes the types of methods that were validated by the EPA OPP method validation program, for both food tolerance enforcement methods and environmental chemistry methods. 

For method tryout, run a control sample and two fortifications from each site. One fortification should be done at the LOQ and the other at the highest expected residue level, perhaps 1000 x LOQ. If the recoveries are within the acceptable range of 70-120% and there are no interferences, proceed with the method validation. If interferences are present which prevent quantitation of the analyte, try additional cleanup steps with SPE or use a more selective detection method such as liquid chromatography/mass spectrometry (LC/MS). 

Wille K, Vanden Bussche J, Noppe H, De Wulf E, Van Caeter P, Janssen CR, De Brabander HF, Vanhaecke L (2010) A validated analytical method for the determination of perfluorinated compounds in surface-, sea- and sewagewater using liquid chromatography coupled to time-of-flight mass spectrometry. J Chromatogr A 1217(43) 6616-6622... 

High performance liquid chromatography-mass spectrometric methods Nitin et al. [75] developed and validated a sensitive and selective liquid chromatography-tandem mass spectrometric method (LC MS MS) for the simultaneous estimation of bulaquine and its metabolites primaquine in monkey plasma. The mobile phase consisted of acetonitrile ammonium acetate buffer (20 mM, pH 6) (50 50, v/v) at a flow rate of 1 mL/min. The chromatographic separations were achieved on two Spheri cyano columns (5 pm, 30 cm x 4.6 mm), connected in... 

The analyses of illicit drugs and metabolites in the sampled waters were performed following a previously described and validated fully automated method based on on-line solid phase extraction-liquid chromatography-tandem mass spectrometry (on-line SPE-LC-MS/MS) [19]. 

Chu, I. et al. 2000. Validation of higher throughput high-performance liquid chromatography/atmo-spheric pressure chemical ionization tandem mass spectrometry assays to conduct cytochrome P450s... 

King, R.C. et al. 2002. Description and validation of a staggered parallel high performance liquid chromatography system for good laboratory practice level quantitative analysis by liquid chromatography/tandem mass spectrometry. Rapid Commun. Mass Spectrom. 16 43. 

Keevil, B.G. et al. 2004. Validation of an assay for voriconazole in serum samples using liquid chromatography-tandem mass spectrometry. Ther Drug Monit. 26 650. 

Sripalakit, P., Nermhom, P., and Saraphanchotiwitthaya, A. 2006. Validation and pharmacokinetic application of a method for determination of doxazosin in human plasma by high performance liquid chromatography. Biomed Chromatogr. 20 729. 

Miege C, Dugay J, Hennion MC. Optimization, validation and comparison of various extraction techniques for the trace determination of polycyclic aromatic hydrocarbons in sewage sludges by liquid chromatography coupled to diode-array and fluorescence detection. J. Chromatogr. A 2003 995 87-97. 

Many of the more established techniques have been validated through collaborative studies which becomes of greater importance as laboratories seek to become accredited via ISO, EN or related systems where the use of official or well validated methods is mandatory. New instrumental techniques are constantly being reported in the literature but it often requires many years before procedures are introduced, validated and then applied within the food industry. Recent techniques that can be included in this category are capillary electrophoresis and liquid chromatography-mass spectrometry (LC-MS). In time procedures based on these techniques will also become accepted as routine methods and are likely to be adopted by some of the official international bodies like the AOAC International, CEN, ISO, etc. 

Because of their considerable role in human wellfare, carotenoids have been measured not only in plants as primary sources and in human and animal tissues but also in a wide variety of other matrices to find new and economical sources for carotenoids. Thus, the carotenoid accumulation capacity of algae and microalgae have been vigorously investigated. A validated liquid chromatography-electrospray mass spectrometry method have been developed and employed for the separation and quantitative determination of... 

M. Careri, L. Elviri and A. Mangia, Liquid chromatography-electrospray mass spectrometry of /J-carotene and xanthophylls. Validation of the analytical method. J. Chromatogr.A 854 (1999) 233-244. 

F. Calbiani, M. Careri, L. Elviri, A. Mangia, L. Pistara and I. Zagnoni, Development and in-house validation of a liquid chromatography-electrospray-tandem mass spectrometry method for the simultaneous determination of Sudan I, Sudan II, Sudan II and Sudan IV in hot chili products. J. ChromatogrA, 1043 (2004) 123-130. 

Borman, P. J., Chatfield, M. J., Crowley, E. L., Eckers, C., Elder, D. P., Francey, S. W., Laures, A. M-F., Wolff, J-C. Development, validation and transfer into a factory environment of a liquid chromatography tandem mass spectrometry assay for the highly neurotoxic impurity FMTP (4-(4-flurophenyl)-l-methyl-l,2,3,6-tetrahydropyridine) in paroxetine active pharmaceutical ingredient (API). J. Pharm. Biomed. Anal., 48, 2008, 1082-1089. 

In the last twenty years, many of the developed and validated high performance liquid chromatography methods with conventional diode array or fluorescence detectors (DAD, FLD) were improved and substituted by new hyphenation with mass spectrometric instrumentation and/or NMR, especially for the analyses of raw materials derived from Natural sources. The main goal of this coupling is achieved by improvement of selectivity and sensitivity of new instrumental configurations [7], Furthermore, with these configurations it is possible to obtain, in only one analysis, the complete chemical structure elucidation, identification and quantification of targeted compounds. 

Kratzsch C, Peters FT, Kraemer T, Weber AA, Maurer HH. 2003. Screening, library-assisted identification and validated quantification of fifteen neuroleptics and three of their metabolites in plasma by liquid chromatography/ mass spectrometry with atmospheric pressure chemical ionization. J Mass Spectrom 38 283. 

This chapter focuses on approaches to the validation of high-performance liquid chromatography methods based on regulatory guidance documents and accepted industry practices. The information in this chapter gives a brief review of the reasons for performing method validation and the regulations that describe this activity. Individual validation parameters are discussed in relation to the type of method to be validated. Examples of typical validation conditions are presented with references to additional information on individual topics. This chapter was written to help analysts responsible for method validation. 

Hayward, M. J., Hargiss, L. O., Munson, J. L., Mandiyan, S. P. and Wennogle, L. P., Validation of Solubility Measurements Using Ultra-Filtration Liquid Chromatography Mass Spectrometry (UP-LC/MS), American Society for Mass Spectrometry 2000 Conference Abstract, Long Beach, CA, USA, 2000. 

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